Infectious bursal disease virus (IBDV), a member of the Binaviridae family, is the causative agent of a highly immunosuppressive disease in young chickens (Kibenge, F. S. B., et al., J. Gen. Virol., 69, 1757-1775 (1988)). Infectious bursal disease (IBD) or Gumboro disease is characterized by the destruction of lymphoid follicles in the bursa of Fabricius. In a fully susceptible chicken flock of 3-6 weeks of age the clinical disease causes severe immunosuppression, and is responsible for losses due to impaired growth, decreased feed efficiency, and death. Susceptible chickens less than 3 weeks old do not exhibit outward clinical signs of the disease but have a marked infection characterized by gross lesions of the bursa. Damage to the bursa ultimately causes immunodeficiency, which then leads to an increased susceptibility to other etiologic agents (Kibenge, F. S. B., et al., J. Gen. Virol., 69, 1757-1775 (1988)) and interferes with effective vaccination against Newcastle disease, Marek's disease and infectious bronchitis disease viruses.
The virus associated with the symptoms of the disease is called infectious bursal disease virus (IBDV). IBDV is a pathogen of major economic importance to the nation and world's poultry industries. It causes severe immunodeficiency in young chickens by destruction of precursors of antibody-production B cells in the bursa of Fabricius. Immunosuppression causes increased susceptibility to other diseases.
There are two known serotypes of IBDV. Serotype I viruses are pathogenic to chickens whereas serotype 11 viruses infect chickens and turkeys but are nonpathgenic.
IBDV belongs to a group of viruses called Binaviridae which includes other bisegmented RNA viruses such as infectious pancreatic necrosis virus (fish), tellina virus and oyster virus (bivalve mollusks) and drosophila X virus (fruit fly). These viruses all contain high molecular weight (MW) double-stranded RNA genomes.
The capsid of the IBDV virion consists of several structural proteins. As many as nine structural proteins have been reported but there is evidence that some of these may have a precursor-product relationship (Kibenge, F. S. B., et al., J. Gen. Virol., 69, 1757-1775 (1988)). The designation and molecular weights of the viral proteins (VP) are as shown below.
Viral Protein Molecular Weight VP1 90 kDa VP2 41 kDa VP3 32 kDa VP4 28 kDa VP5 (NS) 17 kDa
The IBDV genome consists of two segments of double-stranded (ds)RNA that vary between 2827 (segment B) to 3261 (segment A) nucleotide base pairs (Mundt, E. et al., Virology, 209,10-18 (1995)). The larger segment A encodes a 110-kDa precursor protein in a single large open reading frame (polyprotein ORF) which is cleaved by autoproteolysis to form mature viral proteins VP2, VP3 and VP4 (Hudson, P. J. et al., Nucleic Acids Res., 14, 5001-5012 (1986)). Segment A also encodes VP5, a 17-kDa nonstructural (NS) protein, from a small ORF partly preceding and overlapping the polyprotein ORF. However, this protein is not present in the virion and it is only detected in IBDV-infected cells (Mundt, e., et al., J. Gen. Virol., 76, 437-443, 1995). Therefore, VP5 is designated as NS protein. The smaller segment B encodes VP1, a 97-kDa multifunctional protein with polymerase and capping enzyme activities (Spies, U., et al., Virus Res., 8, 127-140 (1987); Spies, U., et al., J. Gen. Virol., 71, 977-981 (1990)).
It has been demonstrated that the VP2 protein is the major host protective immunogen of IBDV, and that it contains the antigenic region responsible for the induction of neutralizing antibodies (Azad, et al., Virology, 161,145-152 (1987)). The region containing the neutralization site has been shown to be highly conformation-dependent. The VP3 protein has been considered to be a group-specific antigen because it is recognized by monoclonal antibodies directed against it from strains of both serotype I and 11 viruses. The VP4 protein appears to be a virus-coded protease that is involved in the processing of a precursor polyprotein of the VP2, VP3 and VP4 proteins (Jagadish, M. N., et al., J. Virol.,62, 1084-1087, 1988).
The nucleotide sequences for genome segments A and B of various IBDV strains have been published and the complete 5'- and 3'-noncoding sequences of both segments have been determined. The 5'-noncoding region of IBDV segments A and B contain a consensus sequence of 32 nucleotides, whereas the 3'-noncoding terminal sequences of both segments are unrelated, but conserved among IBDV strains of the same serotype (Mundt, E. et al., Virology, 209, 10-18 (1995)). These termini might contain sequences important in packaging and in the regulation of IBDV gene expression, as demonstrated for other dsRNA containing viruses such as mammalian and plant reoviruses, and rotaviruses (Anzola, et al., Proc. Natl. Acad. Sci. USA, 84, 8301-8305 (1987); Zou, S., et al., Virology, 186, 377-388 (1992); Gorziglia, M. I., et al., Proc. Natl. Acad. Sci. USA, 89, 5784-5788 (1992)).
In recent years, a number of infectious animal RNA viruses have been generated from cloned cDNA using transcripts produced by DNA-dependent RNA polymerase (Boyer, J. C., et al., Virology, 198, 415-426 (1994)). For example poliovirus, a plus-stranded RNA virus; influenza virus, a segmented negative-stranded RNA virus; rabies virus, a non-segmented negative-stranded RNA virus; all were recovered from cloned cDNAs of their respective genomes (van der Werf, S., et al., Proc. Natl. Acad. Sci. USA, 83, 2330-2334 (1986); Enami, M., et al., Proc. Natl. Acad. Sci. USA, 87, 3802-3805 (1990); Schnell, M. J., et al., EMBO J., 13, 4195-4205 (1994)). For reovirus, it was shown that transfection of cells with a combination of ssRNA, dsRNA and in vitro translated reovirus products generated infectious reovirus when complemented with a helper virus from a different serotype (Roner, M. R., et al., Virology, 179, 845-852 (1990)).
Recently, one of the present inventors recovered a virus of segmented dsRNA genome from synthetic RNAs only. The reverse genetics system for bimavirus was developed by one of the present inventors who demonstrated that synthetic transcripts of infectious bursal disease virus (IBDV) genome are infectious (Proc. Natl. Acad. Sci. USA, 55:11131-11136, 1996). The present inventors have now determined that the 17 kDa nonstructural (NS)protein encoded by a minor open reading frame of segment A, is not required for viral replication in vitro or in vivo and plays an important role in viral pathogenesis.
Complete nucleotide sequences of the large segment A of various IBDV strains have been determined (Vakharia, V. N., et al., Virus Res., 31, 265-273, 1994). In all cases, the small ORF is invariably present which codes for the 17-kDa NS protein. Recently, it was shown that NS protein is not required for viral replication in vitro (Mundt, E., et al., J. Virol, 71, 5647-5651). However, the function of this protein is still unknown. This protein is highly basic, cysteine-rich and conserved among all serotype I IBDV strains. In chicken anemia virus, another virus causing immunosuppression, an analogous basic, cysteine-rich 14-kDa protein was shown to cause apoptosis, and was implicated in pathogenesis (Noteborn, M. H. M., et al., J. Virol., 68, 346-351, 1994). Since IBDV is also known to induce apoptosis in chicken blood lymphocytes (Vasconcelos, A. C., and Lam, K. M., J. Gen. Virol., 75, 1803-1806,1994), the present inventors speculated that NS protein of IBDV may play a similar role in pathogenesis. Therefore, to study the function of NS protein in viral pathogenesis, the present inventors constructed a cDNA clone of IBDV segment A, in which the initiation codon of the NS gene was mutated to a stop codon. Using the reverse genetics system, a wild-type IBDV was generated, as well as a mutant IBDV that lacked the expression of the NS protein. The properties of the recovered wild-type IBDV and mutant IBDV in cell culture were compared and their pathological function in the natural host evaluated.